PROJECT SUMMARY To understand immunity mechanistically, one must identify its constituent parts: those proteins with non- redundant function in the exercise of any immune process, however we choose to define it. One might assume that most of the essential proteins are known, but they are not. This conclusion is based on growing experience with a new method in mammalian genetics, developed in our laboratory1. By damaging or destroying genes at random with ENU, sequencing the whole exome of every G1 male carrier of these mutations, and pre-genotyping all G3 animals at potential mutation sites prior to phenotypic screening, we are able to determine which mutations cause phenotype in real time. In effect, we now positionally clone instantaneously: when a phenotype is seen, its cause is known. We also know precisely which genes we have altered, what change was made, and how many times the mutation was tested for phenotypic effects in the homozygous state. Over the 20 months prior to submission of this proposal, we applied this method to 30,446 G3 mutant mice from 1,189 pedigrees. Each mutant mouse was subjected to 110 immunological assays testing the immunological effects of 70,144 coding or splicing changes in 17,640 genes. For 1,865 genes, at least one putative null allele was examined three or more times in the homozygous state. We estimate that we have stringently tested 18.4% of all genes in the genome for a necessary role in those immune functions falling within our sphere of interest. In so doing, we have identified many proteins essential for one immunological process or another, some of them known, but most of them previously unrecognized1. This preliminary survey makes it clear that much remains to be learned about phenomena one might regard as well studied. What are the requirements for a T-dependent antibody response? We may think we know, but in fact, many of the essential proteins are still undiscovered. As more than 100 new components of the immune system have been identified through mutagenesis, it is incumbent upon us to understand how at least some of the mutations produce their phenotypic effects. We have chosen to address a subset of genes, within which mutations cause striking phenotypes bearing on adaptive immunity. First, we will probe the basis of allergic responses by studying a set of four genes in which mutations cause exaggerated IgE responses to injected papain and/or ovalbumin/alum immunization. Second, we will examine mutations in three genes not generally known to be associated with immunity that dramatically affect lymphocyte development and/or function. Third, we will study the regulation and function of IgD in light of a mutation that causes a selective hyper-IgD syndrome. We will also continue to mutagenize and screen mice for still other immune defects, and make our data accessible to the scientific community via software developed for this purpose.